2.1. ADJUVANT ACTIVITY AND INFLUENCES ON TOLERANCE BY LT
Oral immunization can lead to loss of systemic reactivity in response to subsequent parenteral injection of the specific antigen (Challacombe and Tomasi, 1980, J. Exp. Med. 152:1459-1472). This phenomenon of immune tolerance after ingestion of antigen has been shown to occur in numerous animal models. A variety of effects may account for this phenomenon, including (a) antigen overload, (b) induction of antigen-specific suppressor T cells, and (c) clonal deletion of antigen-specific T and B cells [recently reviewed by Siskind] (Siskind, 1984, In: Fundamental Immunology (Ed. Paul) Raven Press, New York, pp. 537-558). The abrogation of tolerance (or prevention of its induction) has also been widely examined (Green and Ptak, 1986, Immunol. Today 7:81-87; La Tont, et al., 1982, J. Exp. Med. 142:1573-1578; Suzuki, 1986, Nature 320:451-454). In general, it has been observed that the the ability to influence induction of tolerance depends upon the cellular basis of the state of tolerance. Tolerance can be either complete or partial, and is influenced by antigen dose and characteristics, route of administration, physiological state of the organism, and genetic characteristics of the organism. It has also been shown that tolerance can be terminated or prevented by various manipulations, depending upon the cellular basis of the state of tolerance.
Recently it has been demonstrated that administration of cholera toxin (CT) can abrogate oral tolerance to an unrelated antigen (Elson and Ealding, 1984a, J. Immunol. 132:2736-2741). CT, an 84,000 dalton polymeric protein produced by Vibrio cholerae, consists of two subunits, designated A and B. The 56,000 dalton B subunit binds the toxin to its cell surface receptor, the monosialosylganglioside GM1, and facilitates the penetration of the toxic 28,000 dalton A subunit into the cell. The A subunit catalyzes the ADP-ribosylation of the stimulatory GTP-binding protein (GS) in the adenylate cyclase enzyme complex and this results in increasing intracellular levels of adenosine 3xe2x80x2, 5xe2x80x2-cyclic monophosphate (cAMP) (Finkelstein, 1973, CRC Crit. Rev. Microbiol. 2:553-623; In Mechanisms of Bacterial Toxinology (Ed. Bemheimer) John Wiley and Sons, Inc., New York, pp. 53-84). Some strains of Escherichia coli produce an immunologically and structurally related heat-labile enterotoxin (LT) that has the same subunit organization and arrangement as CT and that works by the same 20 mechanism of action (Clements and Finkelstein, 1979, Infect. Immun. 24:760-769; Clements et al. 1980, Infect. Immun. 29:91-97). Although there are many similarities between CT and LT, there are also immunologic and structural differences between the two toxins (Clements and Finkelstein, 1979; Clements et al., 1980). It should be noted that the relative immunoregulatory potential of LT has not been thoroughly investigated. Recently, a clone of E. coli that produces only the binding subunit of the LT toxin (LT-B) was developed (Clements et al., 1983, Infect. Immun. 40: 653-658; Clements and El-Morshidy, Infect. Immun. 46: 564-569).
Escherichia coli heat-labile enterotoxin and heat-stable enterotoxin have previously been used in compositions that are effective in providing immunologic protection in mammals against acute diarrheal disease caused by enterotoxigenic strains of E. coli. See U.S. Patents 4,053,584; 4,314,993; and 4,411,888 which are incorporated herein by reference; and Frantz et al., 1987, Infect. Immun. 55: 1077-1084, Hussaini and Sawtell, 1986, Dev. Biol. Stand. 64: 261-269.
Although the mechanism for abrogation of tolerance by CT is unknown, it is presumed to result from an alteration of the regulatory environment in the gut associated lymphoid tissue, shifting it toward responsiveness (Elson and Ealding, 1984a). It was previously reported that both subunits of CT (and also of LT) have immunoregulatory potential. The binding subunit can mediate thymocyte proliferation (Spiegel et al., 1985, Science, 230:1285-1287) and act as an efficient carrier for stimulation of anti-hapten IgA responses to unrelated antigens (Elson and Ealding, 1984b, J. Immunol. 133: 2892-2897; McKenzie and Halsey, 1984, J. Immunol. 133: 1808-1824). The A subunit, as mentioned above, stimulates adenylate cyclase activity. CT and LT have been shown to stimulate lipolytic activity of isolated epididymal fat cells from rats (Vaughan et al., 1980, Nature 226:658-659; Greenough, 1970, J. Infect. Dis. 121:5111-5114), elevate cAMP levels in intestinal tissues in vivo (Shafer et al., 1970 Proc. Nat""l. Acad. Sci. USA. 67:851-856), increase delta-4, 3-ketosteroids and induce morphologic alterations in cultured mouse Y-1 adrenal tumor cells (Donta et al., 1973, Nature (New Biol.) 243:246-247; Donta et al., 1974, Science 183:334-336), and to increase accumulation of CAMP and induce morphologic alterations in cultured Chinese hamster ovary cells (Guerrant et al., 1974, Infect. Immun. 10:320-327). Cultured fibroblasts respond with increased cAMP and increased collagen synthesis (Guerrant et al., 1974) cell elongation and adhesion to substrate (Nozawa et al. 1975, Infect. Immun. 12:621-624) as well as by inhibition of nucleotide and amino acid transport and protein synthesis. These toxins have also been shown to stimulate basal adenylate cyclase activity in liver with a concomitant decrease in hepatic glycogen (Hynie et al., 1974, Toxicon 12:173-179), to stimulate adenylate cyclase activity of human embryonic intestinal epithelial cells in culture (Kantor, 1975, J. Infect. Dis. 133:522-532; Kantor et al., 1974, Infect, Immun. 9:1003-1010), and to increase membrane adenylate cyclase activity in mouse thymocytes (Zenser and Metzger 1974, Infect. Immun. 10:503-509) and rat pituitary cells (Rappaport and Grant, 1974, Nature 248:73-75). Presumably, because of the ubiquity of the GM1 ganglioside in cell membranes, CT and LT have been found to have a broad spectrum of activity and, in fact, elevate intracellular levels of cAMP in virtually every mammalian tissue tested (Kantor, 1975). It should be noted that CT has been reported to have limited adjuvant activity (Lycke et al., 1989, J. Immunol. 142:20-27) and is able to abrogate tolerance to unrelated antigens (Elson and Ealding, 1984b).
In an abstract for the 88th Annual Meeting of the American Society for Microbiology, May 8-13, 1988, the oral adjuvant effect of LT was stated based on its capacity to induce serum and mucosal antibodies to the peptide antigens OVA and BSA. LT was not reported in combination with non-living microbial vaccines administered via the oral route at the time of this presentation, ie., LT had not been shown to induce specific, protective mucosal immunity to a pathogenic microorganism. (In this context, it should be noted that inappropriate antibody responses to pathogenic microorganisms can actually enhance their pathogenicity - a phenomenon known as xe2x80x9cimmune enhancementxe2x80x9d and well documented in the disease dengue hemorrhagic fever. Therefore, the capacity of a substance to enhance antibody formation does not demonstrate that the substance will enhance protective immunity capable of assisting the host with clearance of the pathogen.) Nor had the differential toxicity of LT and CT been demonstrated in vivo. The material presented at the American Society for Microbiology Meeting in May of 1988 was subsequently published by Clements, et al., 1988, Vaccine 6:269-276; some of the data in the article regarding induction of immunity to peptides also appears in this Application as filed Jun. 2, 1989.
Accordingly, an object of this invention is an immunological adjuvant for peptide, polysaccharide or non-living microbial vaccines administered via the oral route.
Another object of the invention is a method for inducing protective (sometimes termed adaptive) immunity to pathologic antigens by multiple administrations of adjuvant with appropriate amounts of antigen.
An additional object of the invention is a pharmaceutical composition to induce a protective immune response to a pathogen using a peptide, polysaccharide or non-living microbial vaccine.
A further object of this invention is a pharmaceutical composition for stimulating protective immunity at mucosal surfaces throughout the host.
A further object of this invention is a pharmaceutical composition for stimulating long-lasting protective immunity of mucosal surfaces throughout the host.
A further object of this invention is a pharmaceutical composition for oral priming of the parenteral immune response.
Yet another object of the invention is an adjuvant having low toxicity at adjuvant-effective doses.
Still another object of the invention is the alteration of extant immunity manifested as allergic responses at mucosal surfaces, including the intestine and the respiratory tract.
These and additional objects of the invention are accomplished by the use of the heat-labile enterotoxin (LT) of E. coli as an immunological adjuvant for enhancing an animal""s (host) immune response. In particular, LT potentiates the production of antigen-specific serum IgG and mucosal IgA as well as cellular immune responses following multiple administrations via the oral route simultaneously with antigen.
The antigen may consist of a peptide or polysaccharide component of a microorganism containing epitopes important in protective immunity to that microorganism, or of a non-living microorganism or extract of that microorganism containing epitopes essential for protective immunity to the microorganism. Alternatively, the antigen may consist of substances containing epitopes shared by substances (allergens) to which the host has previously established an atopic immune response (generally mediated by IgE antibodies) evoking allergic manifestations at mucosal surfaces.
It is apparent to someone who is skilled in the art that this invention will be useful for any specific antigen where a specific neutralizing antibody response would be useful in ablating the physiological or disease state associated with that antigen.